MEDIATION OF CHEMOATTRACTANT-INDUCED CHANGES IN [CA2+](I) AND CELL-SHAPE, POLARITY, AND LOCOMOTION BY INSP(3), DAG, AND PROTEIN-KINASE-C IN NEWT EOSINOPHILS

During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+](i) from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+](i) and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+](i) increased rapidly from less than or equal to 100 nM to greater than or equal to 400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+](i) fluctuated around a mean value of similar to 200 nM. The initial Ca2+ spike was insensitive acutely to removal of extracellular Ca2+ but was abolished by treatments expected to deplete internal Ca2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP(3). Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lamellipod activity and suppressed the Ca2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca2+ spike on stimulation. Although cell flattening and lamellipod activity were induced by chemoattractant when the normal Ca2+ response was blocked, cells failed to polarize and crawl, indicating that Ca2+ homeostasis is required for these processes. We conclude that InsP(3) acting on Ca2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+](i), which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP(3) and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.